TWI736173B - Mycelium of liquid culture of antrodia camphorata extract, compounds of mycelium of liquid culture of antrodia camphorata extract, and use thereof for treating ischemic stroke - Google Patents

Abstract

The present disclosure provides a mycelium of liquid culture of Antrodia camphorata extract, compounds of the mycelium of liquid culture of Antrodia camphorata extract, and a use thereof for treating ischemic stroke.

Description

Liquid culture extract of Antrodia cinnamomea mycelium, compound of liquid culture extract of Antrodia cinnamomea mycelium and its use for treating ischemic stroke

The present invention relates to an Antrodia cinnamomea extract, an Antrodia cinnamomea extract compound and its use for treating ischemic stroke.

Stroke ranks third among the top ten causes of death in Taiwan, and WHO lists stroke as the "second leading cause of death in the world." In 2005, 5.8 million people died of stroke worldwide, and 6.5 million are expected to be caused by stroke in 2015. die. Taiwan has gradually entered an aging social structure, and aging is an important risk factor for stroke disease. According to the health and medical data of local stroke centers, the risk of stroke for people over 80 is 384 times that of young people under 30.

Ischemic stroke is the rapid development and loss of brain function caused by abnormal blood supply to the brain. In medicine, it is also called a cerebrovascular accident (CVA). It is caused by the destruction of the blood supply to the brain, which makes the cells in the brain unable to obtain sufficient nutrients and oxygen, resulting in damage to the function of the nerves. Stroke can be divided into hemorrhage stroke and ischemia stroke. Generally speaking, ischemic stroke accounts for about 80%, and hemorrhagic stroke accounts for about 20%, and the causes of the two are quite different. Hemorrhagic stroke is cerebral hemorrhage caused by the rupture of a cerebral blood vessel, and the death rate is usually higher. On the other hand, ischemic stroke is cerebral ischemia caused by poor blood circulation or obstruction of the brain. The mortality rate is usually low, but it is easy to cause damage to neurobehavior.

After the U.S. FDA approved venous thrombolytic agent (rt-PA) for the treatment of stroke in 1996, rt-PA is currently the only clinical treatment of ischemic stroke. The mechanism of action of rt-PA is to embolize the group. The blood clot is dissolved to achieve blood recovery, which will increase the probability of cerebral hemorrhage in patients using rt-PA by 10 times. In addition, the disability and injury caused by the body of ischemic stroke patients will cause the family's human care and economic burden, as well as the country's medical and economic burden. Therefore, in order to solve the above-mentioned problems, those skilled in the art urgently need to develop novel medicines with the efficacy of treating stroke to benefit the majority of the people in need, in order to reduce the damage of nerve cells caused by ischemic stroke, thereby increasing the deficiency. The therapeutic effect of bloody stroke.

In view of this, the purpose of the present invention is to provide an Antrodia camphorata extract, which contains ergosta-7,9(11),22-triene-3β-ol (ergosta-7,9(11) , 22-trien-3β-ol, EK100), wherein the Antrodia cinnamomea extract is prepared by extracting the mycelium of an Antrodia cinnamomea with an extraction solvent, the extraction solvent is methanol or ethanol, and the extract is ethyl acetate The ester (or n-hexane) is partitioned and extracted with water, and the ethyl acetate soluble layer (or n-hexane soluble layer) is taken.

Another object of the present invention is to provide an antrodia camphorata (Antrodia camphorata) extract for the preparation of a medicine for the treatment of stroke, wherein the Antrodia camphorata extract contains ergostatin-7,9(11),22-tri Ene-3β-ol (ergosta-7,9(11),22-trien-3β-ol, EK100), the Antrodia cinnamomea extract is prepared by extracting an Antrodia cinnamomea mycelium with an extraction solvent. The extraction The solvent is methanol or ethanol, and the extract is separated with ethyl acetate (or n-hexane) and water, and the ethyl acetate soluble layer (or n-hexane soluble layer) or a combination thereof is taken.

Another object of the present invention is to provide a compound for the preparation of a drug for the treatment of stroke, wherein the compound is ergostatin-7,9(11),22-triene-3β-ol (ergosta- 7,9(11), 22-trien-3β-ol, EK100).

In an embodiment of the present invention, the treatment of stroke is treatment of cerebral apoplexy.

In an embodiment of the present invention, the treatment of cerebral apoplexy is treatment of cerebral ischemic stroke injury.

In an embodiment of the present invention, the treatment of cerebral ischemic stroke injury includes alleviating cerebral infarction and blood brain barrier (BBB) damage after acute ischemic stroke by inhibiting the inflammation and apoptosis of nerve cells.

In an embodiment of the present invention, the treatment of cerebral ischemic stroke injury includes the activation of β-catenin by inhibiting GSK-3 and activating PI3K/Akt signaling to promote endogenous nerve regeneration.

In an embodiment of the present invention, the effective concentration of the Antrodia camphorata liquid fermentation mycelium extract is at least 0.3 g/kg.

In an embodiment of the present invention, the ergostatin-7,9(11),22-triene-3β-ol (EK100) is purified from an Antrodia camphorata extract, and the Antrodia camphorata extract is an extract The solvent is prepared by extracting the mycelium of Antrodia cinnamomea. The extraction solvent is methanol or ethanol, ethyl acetate, n-hexane or a combination thereof.

Another object of the present invention is to provide a compound for the preparation of an agonist that enhances the efficacy of the treatment of stroke, wherein the compound is ergostatin-7,9(11),22-triene-3β -Alcohol (EK100).

In an embodiment of the present invention, the drug for treating stroke is methanol or ethanol, its ethyl acetate soluble part (or n-hexane soluble part) and its main component ergostatin-7,9(11) , 22-trien-3β-ol (ergosta-7, 9(11), 22-trien-3β-ol, EK100) alone or in combination to synthesize tissue plasminogen activator (recombinant tissue plasminogen activator, rt -PA).

In an embodiment of the present invention, the effective concentration of the ergostatin-7,9(11),22-triene-3β-ol (EK100) is at least 60 mg/kg.

In summary, the effect of the Antrodia camphorata extract and its compounds of the present invention is that it can slow down cerebral infarction and blood-brain barrier damage after acute ischemic stroke by inhibiting nerve cell inflammation and apoptosis, by inhibiting GSK-3 and by activating PI3K/ Akt signaling activates β-catenin (β-catenin) to promote endogenous nerve regeneration and protect neurons, so as to treat stroke (especially cerebral ischemic stroke injury) and enhance the efficacy of drugs for treatment of stroke.

The following will further explain the implementation of the present invention. The following examples are used to illustrate the present invention and are not intended to limit the scope of the present invention. Anyone familiar with the art will not depart from the spirit and scope of the present invention. Some changes and modifications can be made. Therefore, the scope of protection of the present invention shall be subject to the scope of the attached patent application.

Figure 1 is a flow chart of the experimental protocol of the present invention, where MCAO represents right middle cerebral artery occlusion.

Figure 2 is a schematic diagram of the effect of the Antrodia camphorata extract and its compounds of the present invention in reducing cerebral infarction after acute ischemic stroke in mice, where S represents the sham operation group; Veh represents the vehicle group; Et(0.3) represents the ethyl acetate layer extraction Substance group 1, namely administration of 0.3g/kg ethyl acetate layer extract; Et(0.6) represents ethyl acetate layer extract group 2, namely administration of 0.6g/kg ethyl acetate layer extract; EK60 represents EK100 group 1, That is, 60mg/kg EK100 is administered; EK120 indicates EK100 group 2, that is, 120mg/kg EK100 is administered; TPA indicates TPA group (rt-PA); TPA/EK60 indicates combination group; * indicates p <0.05.

3A and 3B are schematic diagrams of the effects of the Antrodia camphorata extract and its compounds of the present invention in reducing ischemic stroke injury and blood brain barrier (BBB) damage in mice by inhibiting inflammatory cell occludin, wherein S or Sham Represents the sham operation group; Veh represents the vehicle group; Et (0.3) or EtOAc-AC (0.3) represents the ethyl acetate layer extract group 1; Et (0.6) or EtOAc-AC (0.6) represents the ethyl acetate layer extract Group 2; EK60 means EK100 group 1; EK120 means EK100 group 2; TPA means TPA group (rt-PA); TPA/EK60 means combination group; † means compared with sham operation group, p <0.05; * means with vehicle Compared with the group, p <0.05.

Figures 4A and 4B are schematic diagrams of the effects of the Antrodia camphorata extract and its compounds of the present invention in reducing ischemic stroke injury in mice by inhibiting inflammatory cells p65 NF-κB, where S or Sham represents the sham operation group; Veh represents the vehicle group ; Et(0.3) or EtOAc-AC(0.3) represents ethyl acetate layer extract group 1; Et(0.6) or EtOAc-AC(0.6) represents ethyl acetate layer extract group 2; EK60 represents EK100 group 1; EK120 group 2 represents EK100; TPA TPA represents group (rt-PA); TPA / EK60 represent combinations; † indicates compared with sham group, p <0.05; * represents the group was compared to vehicle, p <0.05;# represents Compared with the TPA group (rt-PA), p <0.05.

Figures 5A and 5B are schematic diagrams of the effects of the Antrodia camphorata extract and its compounds of the present invention in reducing ischemic stroke injury in mice by inhibiting inflammation and apoptosis markers, where S or Sham represents the sham operation group; Veh represents the vehicle group ; Et(0.3) or EtOAc-AC(0.3) represents ethyl acetate layer extract group 1; Et(0.6) or EtOAc-AC(0.6) represents ethyl acetate layer extract group 2; EK60 represents EK100 group 1; EK120 group 2 represents EK100; TPA TPA represents group (rt-PA); TPA / EK60 represent combinations; † indicates compared with sham group, p <0.05; * represents the group was compared to vehicle, p <0.05;# represents Compared with the TPA group (rt-PA), p <0.05.

Figure 6A and Figure 6B show the effect of Antrodia camphorata extract and its compounds in promoting endogenous nerve regeneration after acute ischemic stroke in mice, where S or Sham represents the sham operation group; Veh represents the vehicle group; Et(0.3) or EtOAc -AC(0.3) represents ethyl acetate layer extract group 1; Et(0.6) or EtOAc-AC(0.6) represents ethyl acetate layer extract group 2; EK60 represents EK100 group 1; EK120 represents EK100 group 2; TPA represents TPA group (rt-PA); TPA/EK60 means combination group; † means compared with sham operation group, p <0.05; * means compared with vehicle group, p <0.05;# means compared with TPA group (rt-PA ) Compared with p <0.05.

Figures 7A and 7B show the effect of Antrodia cinnamomea extract and its compounds in promoting nerve regeneration-related protein levels after acute ischemic stroke in mice (at the 24th hour), where S or Sham represents the sham operation group; Veh represents the vehicle group ; Et(0.3) or EtOAc-AC(0.3) represents ethyl acetate layer extract group 1; Et(0.6) or EtOAc-AC(0.6) represents ethyl acetate layer extract group 2; EK60 represents EK100 group 1; EK120 group 2 represents EK100; TPA TPA represents group (rt-PA); TPA / EK60 represent combinations; † indicates compared with sham group, p <0.05; * represents the group was compared to vehicle, p <0.05.

definition

The numerical values used herein are approximate values, and all experimental data are expressed in the range of 20%, preferably in the range of 10%, and most preferably in the range of 5%.

According to the present invention, all experimental data are based on the mean ± SEM (standard error of the mean). One-way analysis of variance (ANOVA) was used, followed by a post-hoc Student-Newman-Keuls (SNK) t test to analyze the data for multiple comparisons. For all pairs of multiple comparisons, the Log-Rank test was used, and then the Holm-Sidak method was used to evaluate the survival rate. A value of p <0.05 is considered to be statistically significant.

According to the present invention, Antrodia camphorata (Antrodia camphorata), also known as Antrodia camphorata, Antrodia camphorata, Wild rice, and Shenming mushroom, is a medicinal fungus, belonging to the genus Fomitopsidaceae ( Antrodia ). Fungus, only native to Taiwan.

The "effective concentration" mentioned herein refers to the amount of medicine needed to directly treat individuals suffering from stroke. The effective concentration may be different depending on the biological species or individual differences to be treated, but the effective concentration can be determined experimentally by, for example, a concentration escalation test.

As used herein, "treating" or "treatment" means alleviating, reducing, ameliorating, relieving or controlling a disease ) Or one or more clinical signs of disorder, as well as the severity of lowering, stopping or reversing a condition or symptom being treated The progression of severity.

According to the present invention, the medicine can be manufactured into a dosage form suitable for parenterally or orally administration by using techniques well known to those skilled in the art. This includes, but not Limited to: injection (for example, sterile aqueous solution or dispersion), sterile powder, tablet, troche, lozenge Lozenge, pill, capsule, dispersible powder or granule, solution, suspension, emulsion, syrup, elixir ), slurry and the like.

The medicine according to the present invention can be administered by a parenteral route selected from the group consisting of: intraperitoneal injection, subcutaneous injection, intramuscular injection (intramuscular injection) and intravenous injection.

The pharmaceutical product according to the present invention may contain a pharmaceutically acceptable carrier widely used in pharmaceutical manufacturing technology. For example, the pharmaceutically acceptable carrier may include one or more agents selected from the group consisting of solvents, emulsifiers, suspending agents, decomposing agents (decomposer), binding agent, excipient, stabilizing agent, chelating agent, diluent, gelling agent, preservative ), lubricant (lubricant), absorption delaying agent (absorption delaying agent), liposome (liposome) and the like. The selection and quantity of these reagents fall within the scope of professionalism and routine techniques of those who are familiar with this technique.

According to the present invention, the pharmaceutically acceptable carrier contains a solvent selected from the group consisting of water, normal saline, and phosphate buffered saline (PBS) , Sugar-containing solutions, aqueous solutions containing alcohol (aqueous solution containing alcohol), and combinations thereof.

According to the present invention, medicines can be used as food additives, which are added during the preparation of raw materials by conventional methods, or added during the production of food, and are formulated with any edible material for supply. Food products consumed by humans and non-human animals.

According to the present invention, the types of food products include, but are not limited to: beverages, fermented foods, bakery products, health foods, and dietary supplements.

Example 1. Preparation of Antrodia camphorata (Antrodia camphorata) extract and its compound and evaluation of its effectiveness in the treatment of brain infarction

First, the preparation process of the Antrodia camphorata extract and its compounds of the present invention is as follows: Refer to YYShao, CCChen, HYWang, HLChiu, THHseu and YHKuo, Chemical constituents of Antrodia camphorata submerged whole broth, Nat. Prod. Res. , 2008, 22 , 1151 The method described in the 1157 literature carries out the preparation of Antrodia cinnamomea extract and its compounds. In short, 4.782 kg of Antrodia camphorata (Antrodia camphorata) mycelium powder is prepared, and then extracted with methanol (MeOH) or ethanol (Ethanol, EtOH) to obtain 1681.28 g of methanol extract (or ethanol extract). Next, take the methanol extract (or ethanol extract) and use the liquid phase partitioning treatment of ethyl acetate (EtOAc) and water to form an ethyl acetate layer extract and an aqueous layer extract. After that, The ethyl acetate layer was collected, and then the aqueous layer was removed. An example of the present invention is to obtain 282.5 mg of ethyl acetate layer extract from 1.0 g of methanol extract, and chromatograph it on silica gel with 10% ethyl acetate (in hexane), and then recrystallize to obtain The purified compound: ergosta-7,9(11), 22-trien-3β-ol (ergosta-7,9(11), 22-trien-3β-ol, hereinafter referred to as EK100), with The following chemical formula (I):

The methanol or ethanol extract, the ethyl acetate layer extract, and EK100 were dissolved in dimethyl sulfide (DMSO) to separate stock solutions of 50 mg/ml, and stored at -20°C. The synthetic tissue plasminogen activator (rt-PA) (the only FDA approved drug for ischemic stroke) used in an embodiment of the present invention was purchased from a pharmaceutical company (Boehringer Ingelheim GmbH, Ingelheim am Rhein, Germany). Physiological saline containing 0.1% DMSO was used as a reference reagent or vehicle control group.

Then, induce and prepare the acute cerebral ischemic stroke injury mouse model, the process is as follows: all animal procedures and procedures are in accordance with the guide for the care and use of laboratory animals (The Guide for the Care and Use of Laboratory Animals) Animals) (U.S. National Research Council, 2011) and reviewed and approved by the Animal Research Committee of the National Research Institute of Chinese Medicine of the Ministry of Health and Welfare (Approval Number: NRICM-IACUC -106-912-1). Use a mixture of zolazepam (40mg/kg, ip) and xylazine hydrochloride (10mg/kg, ip) to anesthetize male ICR mice weighing 28~30g (National Institute of Experimental Research, Taipei, Taiwan) Laboratory Animal Center). According to the recommendations of the Stroke Therapy Academic Industry Roundtable (STAIR), it induced acute cerebral ischemic stroke injury in mice. In short, a heat-blunted nylon monofilament surgical suture (about 100μm in diameter) coated with silicone is used for transient focal cerebral ischemia. , The surgical suture was introduced into the exposed external carotid artery, advanced to the internal carotid artery, and wedged into the Willie’s ring to block the origin of the right middle cerebral artery (MCA), thereby blocking the blood flow of MCA Flow, which can be confirmed by intracranial Doppler (transcranial Doppler) examination to confirm blood flow has stopped. Place the filament under anesthesia for 40 minutes. After 40 minutes of right middle cerebral artery occlusion (MCA occlusion, MCAO), the suture was withdrawn for reperfusion. All treatments were given 2 hours after reperfusion (see Figure 1). In order to reduce treatment bias, researchers unrelated to this study randomly assigned mice to vehicle control group or reagent (methanol extract, ethanol extract, ethyl acetate layer extract, EK100 or rt-PA) treatment group. After the above operation, the surgical incision was closed and the mice were allowed to recover from anesthesia under a heating lamp to ensure that the body temperature was maintained at 36.0~37.0°C during the recovery process. This surgical procedure resulted in reproducible infarcts, the size and distribution of which were similar to other infarct volumes reported using similar transient MCAO techniques.

Blood samples collected from the femoral artery before MCAO and 30 minutes after reperfusion were used immediately for arterial blood gas analysis. The mice were randomly divided into the following groups: (1) Sham operation group without MCAO (Sham, n=30); (2) 2 hours after MCAO, once a day, once a day with a 0.3g/kg methanol extract, orally) Group (methanol extract group) (stroke + methanol extract, n=10); (3 and 4) two ethyl acetate layer extracts (0.3g/kg or 0.6g/kg, 2 hours after MCAO, every day One time, oral) treatment group (ethyl acetate layer extract group 1 (0.3g/kg ethyl acetate layer extract) and ethyl acetate layer extract group 2 (0.6g/kg ethyl acetate layer extract)), n=30; (5 and 6) two EK100 (60mg/kg or 120mg/kg) treatment groups, once a day, orally, 2 hours after MCAO (EK100 group 1 (60mg/kg EK100) and EK100 group 2 (120mg/kg) kg EK100)); (7) 1 rt-PA (rt-PA dose is 10mg/kg, 2 hours after MCAO, intravenous injection once) MCAO treatment group (stroke + rt-PA, n=30) ( TPA group (rt-PA)); (8) 1 rt-PA+EK100 (rt-PA is 10mg/kg, 2 hours after MCAO, an intravenous injection of rt-PA plus EK100 is taken orally at 60mg/kg ) (Combination group); (9) group treated with MCAO and vehicle (normal saline containing 0.1% DMSO) (stroke + vehicle, n=30) (vehicle group). The experimental program is shown in Figure 1.

In order to confirm the ability of methanol extract (0.3g/kg) or ethanol extract and ethyl acetate layer extract (0.3g and 0.6g/kg) of Antrodia cinnamomea to improve ischemic stroke brain damage and the best protective dose of EK100 ( 30~120mg/kg, orally), in order to clarify the mechanism of acute ischemic stroke injury model, and evaluate the animal survival rate (within 2 days after the stroke is induced). Almost 80% of the mice (vehicle group) treated with vehicle (normal saline containing 0.1% DMSO) died within 2 days after stroke. Conversely, methanol extract group or ethanol extract group, ethyl acetate layer extract group 1, ethyl acetate layer extract group 2, EK100 group 1, EK100 group 2, TPA group (rt-PA), and combination group Has a higher survival rate. For mice treated with ethyl acetate layer extract group 2, EK100 group 1, EK100 group 2, and combination group, this difference was particularly significant on day 1 (D1) after stroke (Table 1, p <0.05).

Twenty-four hours after the stroke was induced, the mice were also examined for neurological deficits (measured by the decrease in the movement distance of the track). Compared with mice in the sham operation group, in the mice in the vehicle group, stroke surgery induced extremely severe neurological deficits, such as a significant reduction in the distance of movement (in m) (Table 1, vehicle group vs. Sham operation group, p<0.05). Methanol extract group, ethanol extract group, ethyl acetate layer extract group 1, ethyl acetate layer extract group 2, EK100 group 1, and EK100 group 2, but not rt-PA treatment, all significantly improved after stroke injury This resulted in a decrease in the movement distance of the whereabouts (Table 1, p <0.05). However, the combined group can significantly improve the movement distance of the track compared with the TPA group alone (rt-PA) (Table 1, p <0.05).

Example 2. Evaluation of the effectiveness of Antrodia camphorata extract and its compounds in reducing cerebral infarction and BBB damage after acute ischemic stroke in mice by inhibiting inflammation and apoptosis

The 2,3,5-triphenyltetrazolium chloride (TTC) staining procedure used in the following experiments is as follows: Twenty-four hours after reperfusion, rapid decapitation under deep anesthesia Sacrifice mice. Quickly take out the whole brain and cut into 1mm thick crown Sections were stained with TTC (Sigma-Aldrich, USA). All brain slices were photographed to determine the infarct volume and confirm the edema.

Acute ischemic stroke can cause severe cerebral infarction of the ischemic cerebral hemisphere and severe damage to the brain tissue. The infarct volume is about 62±5mm ³ (Figure 2, stroke + vehicle, TTC staining), and intense inflammation (p65NFκB staining (red) ), which is an inflammatory marker) (Figures 3A and 4A), it is manifested around all infarct areas of the stroke cortex and the dentate gyrus (in the stroke group) with obvious blood-brain barrier (BBB) leakage (Figure 3A and 3B, stroke group, lack of occludin staining (green), and very significant brain apoptosis (Figures 5A and 5B, stroke group, caspase 3 staining (red) ). On the contrary, using the ethyl acetate layer extract of Antrodia cinnamomea (0.3 and 0.6kg/kg, oral) and EK100 (60~120mg/kg, oral) to treat stroke-injured mice can reduce mortality and neurological deficits, but also It can significantly reduce the severity of cerebral infarction (decrease in TTC staining) (Figure 2, p <0.05), BBB leakage (lack of occluding staining) (Figure 3B, p <0.05), and reduce inflammation (p65NFκB staining) ) (Figure 4B, p <0.05) and reduced brain apoptotic cells (apoptotic protease 3) (Figure 5B, p <0.05). Treatment with rt-PA alone is not enough to protect brain tissue from these ischemic brain injuries (cerebral infarction, BBB injury, inflammation, and brain cell apoptosis) (compared to the stroke group, Figure 2 to Figure 5B, p >0.05), in addition to reducing p65NFκB staining (Figure 4A and Figure 4B, compared with the stroke group, p <0.05), however, it is impressive that compared with the use of rt-PA or EK100 (60mg/kg) alone In contrast, the combination of EK100 (60 mg/kg) and rt-PA can further enhance the protective activity (Figure 2 to Figure 5B, p <0.05).

Example 3. Antrodia camphorata extract and its compounds promote endogenous nerve regeneration by inhibiting GSK-3 and activating β-catenin by activating PI3K/Akt signaling after acute ischemic stroke in mice Effectiveness evaluation

Acute ischemic stroke can severely damage the endogenous neurogenesis around the hippocampal dentate gyrus. This example investigates whether the ethyl acetate layer extract of Antrodia cinnamomea and EK100 treatment can promote endogenous nerve regeneration 24 hours after ischemic stroke injury, using doublecortin (DCX) as an indicator to clarify endogenous nerve regeneration . Compared with the sham operation group, the expression level of DCX staining in mice treated with vehicle after ischemic stroke injury was significantly reduced (orange, Figure 6A, stroke + vehicle in the dentate gyrus (Veh)) (Figure 6A and Figure 6B, stroke + Veh vs. sham operation group (Sham, S), p <0.05). It is worth noting that treatment with ethyl acetate layer extract of Antrodia cinnamomea and EK100 instead of rt-PA can strongly upregulate the performance of DCX. Compared with the vehicle group (Figure 6B, p <0.05), it is almost the same as the chain protein (red ) Co-localization of staining (Figure 6A and 6B). In addition, compared with the vehicle-only group (Figure 6B, p <0.05) or the rt-PA-only group, the co-treatment group of EK100 (60mg/kg) and rt-PA (combination group) was significantly upregulated The performance of DCX and the performance of chain protein (Figure 6B, p <0.05).

In order to determine whether the nerve regeneration promotion signal (GSK-3 inhibition and β-catenin activation) is involved in the neuroprotection induced by the ethyl acetate layer extract of Antrodia cinnamomea and EK100, Western blot analysis was used to detect PI3K/Akt-related signal transmission pathways Protects the ethyl acetate extract of Antrodia cinnamomea and EK100. The results showed that stroke injury significantly reduced the expression level of Akt phosphorylation/activation on Ser473 (pAkt) residues, and stroke also significantly reduced the expression of SpGSK-3 (GSK-3 inhibition) and β-catenin and decreased β -The performance of the downstream protein Bcl-2 (Figure 7B, vehicle group compared with sham operation group, p <0.05), in which Ser473 is the amino acid of Akt. Serine at position 473 is phosphorylated , Which means Akt is phosphorylated and activated (pAkt). Treatment with ethyl acetate layer extract of Antrodia cinnamomea (0.6g/kg) and EK100 (60 and 120mg/kg) significantly enhanced the performance level of Bcl-2, possibly through activation of PI3K/Akt-dependent GSK-3 inhibition (SpGSK -3 performance increase) and β-catenin up-regulation (Figure 7A to Figure 7B, p <0.05), and the ethyl acetate layer extract of Antrodia camphorata (0.6g/kg) or EK100 (120mg/kg) was used alone The non-stroke mice did not significantly change the expression level of these proteins (Figure 7B).

Example 4. Antrodia cinnamomea extract and its compounds in the oxygen and glucose deprivation (oxygen and glucose deprivation, OGD) induced neuronal apoptosis and LPS-mediated microglia cell (microglial cell) activation of neuron protection and Evaluation of the effectiveness of anti-inflammatory

In order to determine whether the ethyl acetate layer extract of Antrodia camphorata and EK100 can prevent the apoptosis of nerve cells induced by simultaneous oxygen and glucose deprivation (OGD) and show anti-inflammatory activity in the cell model, the OGD-induced neuroblastic tumor Neuro 2A was performed Cells (from ATCC, CCL-131) apoptosis and LPS-mediated microglia cell nitric oxide (NO) production (ie BV2/NO shown in Table 2, where BV2 stands for BV2 cell, which is a brain microglia cell (microglia cell) strain). As shown in Table 2, the ethyl acetate layer extract of Antrodia cinnamomea and EK100 of the present invention have anti-apoptotic activity against OGD-induced neuronal damage, with IC ₅₀ <10 μg/ml and 40 μM, respectively. The positive control group AR-a014418 (a GSK-3 inhibitor) showed a strong anti-apoptotic effect in this model, with an IC _{50 of} about 10 μM. In addition, the ethyl acetate layer extract of Antrodia camphorata of the present invention has a significant anti-inflammatory effect on LPS-induced NO production in microglia cells, with IC ₅₀ of 9±1 μg/ml and 18±2 μM, respectively. They are almost as effective as NFκB inhibitors (PDTC).

The process of OGD-induced neuronal apoptosis is as follows: Neuro 2A cells are grown in Dulbecco's Modified Eagle Medium (DMEM) at 37°C and supplemented with 10% (by volume) fetal bovine serum (FBS) and 5 % _{Penicillin/streptomycin in CO 2} and 95% humid environment. In the presence of the test reagent, the cells (5×10 ⁵ cells) were subjected to oxygen and glucose deprivation (OGD) for 8 hours, and then perfused for 16 hours. Remove the medium and use HEPES buffered salt solution (HBSS, containing (in mM): 137 NaCl, 5 KCl, 20 HEPES, 1.4 CaCl ₂ , 3 NaHCO ₃ , 0.6 Na ₂ HPO ₄ , 0.4 KH ₂ PO ₄ at pH 7.4 Washing), wash until all the medium containing glucose is removed. Then, start OGD by changing the medium to serum- and glucose-free DMEM (Gibco Laboratories, Grand Island, NY, USA), first _{bubbling it with a mixture of 5% CO 2} and the rest of N ₂ for 20 minutes to remove O ₂ . The culture was then transferred to an incubator (Astec, Fukuoka, Japan) immediately, the low oxygen humidified incubator having a 5% CO2,1% O2 and the balance N _2, and kept at 37 ℃. After hypoxia, the OGD in the culture was stopped by replenishing DMEM with glucose, and then transferred back to an incubator in a normal environment _{with 5% CO 2 in the air for 16 hours.}

In order to check whether the ethyl acetate layer extract of Antrodia cinnamomea alone or EK100 showed antioxidant capacity, a DPPH free radical scavenging test was carried out.

The measurement process of DPPH radical scavenging ability is as follows: the reagent is diluted in methanol to concentrations of 5, 20, and 50 μM. A DPPH solution (200 μl; final concentration of 200 μM in ethanol) was added to 10 μl of each diluted sample in a 96-well microplate, and the resulting solution was allowed to react for 30 minutes at ambient temperature in the dark. The absorbance of DPPH radicals at 517 nm was measured with a microplate spectrophotometer. The free radical scavenging ability is expressed as OD ₅₁₇ (△OD ₅₁₇ ), including the antioxidant trolox as a reference reagent.

Compared with trolox (a vitamin E analog), neither the ethyl acetate extract of Antrodia camphorata nor EK100 showed free radical scavenging activity, while trolox showed DPPH free radical scavenging activity with IC ₅₀ of 25±1μM.

Using an experimental cerebral ischemic stroke rat model, the present invention proves the effect of Antrodia camphorata extract and its active ingredient EK100 on neuroprotection and promoting nerve regeneration after ischemia-reperfusion, and also determines the molecular signal transmission mechanism. The results showed that 2 hours after the induction of acute ischemic stroke, treatment with the ethyl acetate layer extract of Antrodia cinnamomea (0.6g/kg) and EK100 (60-120mg/kg, orally) once a day significantly improved the survival rate and whereabouts. Mobility activity (day 1 after stroke). The combination of low dose (60mg/kg) of EK100 and rt-PA (10mg/kg, i.v.) can enhance the protective activity of rt-PA. Ethyl acetate layer extract and EK100 induced inflammation and apoptosis of brain damage markers were highly correlated with GSK-3 inhibition and β-catenin up-regulation. Specifically, the ethyl acetate layer extract of Antrodia cinnamomea and EK100 down-regulate NF-κB and caspase 3 (caspase 3), and promote nerve regeneration-related protein (Dicortin DCX) and neuroprotective protein (Bcl-2) Performance level. The present invention confirms that the activation of GSK-3/β-chain protein is inhibited by PI3K/Akt signal transmission, and the ethyl acetate layer extract of Antrodia cinnamomea and its active ingredient EK100 have neuroprotective and nerve regeneration promoting effects after acute ischemic stroke injury. .

In the present invention, it is observed that the number of cells involved in endogenous nerve regeneration (DCX positive cells) near the hippocampal dentate gyrus of mice with ischemic stroke is significantly reduced. Nevertheless, using only the ethyl acetate layer extract of Antrodia cinnamomea (0.3 and 0.6g/kg) and EK100 (60 and 120mg/kg), but not using rt-PA alone, the treatment can significantly increase the number of DCX-positive cells. DCX Positive cells are the producers of neuroblasts/neuronal progenitor cells within 24 hours after cerebral ischemic stroke. In addition, the combination of low-dose (60mg/kg) EK100 and rt-PA can promote the expression level of DCX-positive cells. This promotion is likely to occur through the activation of PI3K/Akt, and the activation of PI3K/Akt will cause GSK-3 phosphorylation to inhibit and β-catenin activation, thereby upregulating its downstream cytoprotective proteins (such as Bcl- 2). However, in the present invention, using the ethyl acetate layer extract of Antrodia camphorata and EK100 alone in mice without ischemic stroke injury did not significantly activate the PI3K/Akt pathway to up-regulate the expression level of DCX-positive cells. The present invention found that the protective effect of the ethyl acetate layer extract of Antrodia camphorata or EK100 (rather than rt-PA alone) simultaneously down-regulated the expression of apoptotic protease 3 and p65NF-κB. In addition, using an in vitro model, it was further observed that the ethyl acetate layer extract of Antrodia camphorata and EK100 showed strong anti-apoptotic effects (IC ₅₀ at <10 μg/ml and 40 μM, respectively) and anti-inflammatory (IC ₅₀ at 9 And 18μM) effect.

In summary, the Antrodia camphorata extract and its compounds of the present invention can slow down cerebral infarction and blood-brain barrier damage after acute ischemic stroke by inhibiting inflammation and apoptosis, and activate β by inhibiting GSK-3 and by activating PI3K/Akt signaling. -Catenin promotes endogenous nerve regeneration and protects neurons to achieve the effect of treating stroke (especially cerebral ischemic stroke injury) and enhancing the effectiveness of the treatment of stroke drugs.

The above descriptions are merely illustrative and not restrictive. Any equivalent modifications or alterations that do not depart from the spirit and scope of the present invention should be included in the scope of the appended patent application.

Claims (6)

The use of a compound for the preparation of a medicine for the treatment of stroke, wherein the compound is ergosta-7,9(11),22-triene-3β-ol (ergosta-7,9(11),22 -trien-3β-ol, EK100), wherein the treatment of stroke is the treatment of cerebral ischemic stroke injury, and the treatment of cerebral ischemic stroke injury includes slowing down cerebral infarction and blood brain after acute ischemic stroke by inhibiting inflammation and apoptosis Barrier (blood brain barrier, BBB) damage, and activation of β-catenin (β-catenin) by inhibiting GSK-3 and activating PI3K/Akt signaling to promote endogenous nerve regeneration. The use as described in item 1 of the scope of patent application, wherein the ergostatin-7,9(11),22-triene-3β-ol (EK100) is purified from an Antrodia cinnamomea extract, and the Antrodia cinnamomea extract It is prepared by extracting the mycelium of Antrodia cinnamomea with an extraction solvent, and the extraction solvent is methanol, ethanol, ethyl acetate, n-hexane or a combination thereof. The use as described in item 1 of the scope of the patent application, wherein the effective concentration of the ergostatin-7,9(11),22-triene-3β-ol (EK100) is at least 60 mg/kg. The use of a compound for preparing an agonist that enhances the effectiveness of a drug for treating stroke, wherein the compound is ergostatin-7,9(11),22-triene-3β-ol (EK100), wherein the The treatment of stroke drugs has the effect of treating cerebral ischemic stroke injury. The treatment of cerebral ischemic stroke injury includes slowing down cerebral infarction and blood brain barrier (BBB) damage after acute ischemic stroke by inhibiting inflammation and apoptosis , And by inhibiting GSK-3 and activating β-catenin by activating PI3K/Akt signaling to promote endogenous nerve regeneration. The use as described in item 4 of the scope of patent application, wherein the drug for treating stroke is synthetic tissue plasminogen activator (rt-PA). The use according to item 4 of the scope of patent application, wherein the effective concentration of the ergostatin-7,9(11),22-triene-3β-ol (EK100) is at least 60mg/kg.

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